Nε-(Carboxymethyl)lysine Modification of Elastin Alters Its Biological Properties: Implications for the Accumulation of Abnormal Elastic Fibers in Actinic Elastosis

Accumulation of degenerated elastic fibers in the sun-exposed skin designated as actinic elastosis is a histological hallmark of photodamaged skin. Previous studies have indicated that the elastic fibers of actinic elastosis interact with lysozyme and are modified by Nε-(carboxymethyl)lysine (CML), one of the major advanced glycation end products (AGEs). We studied here how CML modification of elastin is involved in the pathogenesis of actinic elastosis. The CML-modified insoluble elastin became resistant to neutrophil elastase digestion, which was reversed by treatment with aminoguanidine, a potent inhibitor of AGE formation. In a temperature-dependent aggregation assay, CML-modified elastin rapidly formed self-aggregates, the size of which was larger than unmodified elastin. The elastic fiber sheets prepared from CML-modified α-elastin showed 3D wider diameter, tortuous appearance, and decreased elasticity on tensile tests. The CML-modified α-elastin, but not unmodified α-elastin, was found to bind to lysozyme in vitro, supporting the immunohistochemical findings that the antibodies for lysozyme and CML reacted simultaneously with the elastic fibers of actinic elastosis and UV-irradiated skin. The glycated elastin is likely to cause the accumulation of abnormally aggregated elastic fibers and unusual interaction with lysozyme in actinic elastosis

The Receptor for Advanced Glycation End Products Is Highly Expressed in the Skin and Upregulated by Advanced Glycation End Products and Tumor Necrosis Factor-Alpha

Advanced glycation end products (AGEs) form non-enzymatically from reactions of proteins with reducing sugars. In the skin, AGEs were reported to accumulate in dermal elastin and collagens and to interact nonspecifically with the cell membrane of dermal fibroblasts. Therefore, AGEs may influence the process of skin aging. We investigated the presence of the AGE receptor RAGE in skin and the influence of AGEs on receptor expression and the formation of extracellular matrix (ECM). Sections of sun-protected and sun-exposed skin were analyzed with monoclonal antibodies against (RAGE), heat-shock protein 47, factor XIIIa, CD31, and CD45. RAGE was mainly expressed in fibroblasts, dendrocytes, and keratinocytes and to a minor extent in endothelial and mononuclear cells. Human foreskin fibroblasts (HFFs) highly expressed RAGE on the protein and mRNA level when analyzed by quantitative Western blotting and real-time PCR. Incubation of HFFs with the specific RAGE ligand Nε-(carboxymethyl)lysine-modified BSA (CML-BSA) and tumor necrosis factor-alpha resulted in significant upregulation of RAGE expression. CML-BSA induced a mildly profibrogenic pattern, increasing connective tissue growth factor, transforming growth factor-beta (TGF-β)1, and procollagen-α1(I) mRNA, whereas expression of matrix metalloproteinase (MMP)-1, −2, −3, and −12 was unaffected. We conclude that in HFFs, AGE–RAGE interactions may influence the process of skin aging through mild stimulation of ECM gene expression

Photo-Enhanced Modification of Human Skin Elastin in Actinic Elastosis by N∈-(Carboxymethyl)lysine, One of the Glycoxidation Products of the Maillard Reaction

Long-term incubation of proteins with glucose leads to the formation of advanced glycation end products (AGEs), which are characterized by fluorescence, brown color, and cross-linking. Formation of AGEs in vitro requires oxygen and is dependent on transition metal-catalyzed oxidation of glucose or Amadori products. AGEs are thought to be involved in aging and age-enhanced diseases such as diabetic complications, atherosclerosis, dialysis-related amyloidosis, and Alzheimer's disease. Chronic exposure of the skin to sunlight induces hyperplasia of the elastic tissue in the upper dermis known as actinic elastosis. Herein we used a monoclonal anti-AGE antibody (6D12) whose epitope is N∈-(carboxymethyl)lysine (CML), one of the glycoxidation products of AGEs, and demonstrated that the lesions of actinic elastosis were modified by CML. Further immunohistochenilcal and immunoelectron microscopic examination with 6D12 demonstrated CML accumulates predominantly in elastic fibers especially in the amorphous electron-dense materials corresponding to photo-induced degenerated area rather than the electron-lucent region. Immunochemical analyses with enzyme-linked immunosorbent assay (ELISA) of elastase-soluble fractions demonstrated that the CML levels of the sun-exposed area were significantly higher than those of the sun-unexposed area. We conclude that ultraviolet-induced oxidation may accelerate CML formation in actinic elastosis of photoaged skin

Age- and sun exposure-dependent differences in 8-hydroxy-2'-deoxyguanosine and Nε-(carboxymethyl)lysine in human epidermis

Aging and exposure to sunlight are two major factors in the deterioration of skin function. In this study, thirty-six fixed human skin samples from sun-exposed and unexposed areas from young and old individuals were used to evaluate the localization of oxidative stress according to levels and distribution of 8-hydroxy-2'-deoxyguanosine and Nε-(carboxymethyl)lysine in samples using immunohistochemistry. In the epidermis of the young, negligible amounts of 8-hydroxy-2'-deoxyguanosine and Nε-(carboxymethyl)lysine were detected in unexposed areas, whereas nuclear 8-hydroxy-2'-deoxyguanosine and cytoplasmic Nε-(carboxymethyl)lysine were increased in the lower epidermis in sun-exposed areas. In contrast, the aged presented prominent nuclear 8-hydroxy-2'-deoxyguanosine and nuclear Nε-(carboxymethyl)lysine in the epidermis of unexposed areas, concomitant with dermal increase in Nε-(carboxymethyl)lysine. However, the immunostaining of 8-hydroxy-2'-deoxyguanosine and Nε-(carboxymethyl)lysine revealed a decrease in the epidermis of sun-exposed areas in the aged. These results suggest an age-dependent difference in the adaptation and protective mechanisms of the epidermis against sunlight-associated oxidative stress, thus necessitating distinct standards for evaluation in each age group. Further investigation is warranted to elucidate underlying molecular mechanisms

Immunohistochemical localisation of d-β-aspartic acid in pingueculae

Background: D-β-Aspartic acid residues, which are biologically uncommon, have been reported to accumulate in various proteins of the living body with age. In the present study, D-β-aspartic acid-containing proteins were found to be localised in pingueculae, which represent one of the most prominent age-related ocular changes.Methods: Surgical specimens of conjunctivae with or without pingueculae were obtained from eight patients. Immunohistochemical localisation of D-β-aspartic acid-containing proteins was performed using a polyclonal antibody against D-β-aspartic acid-containing peptides.Results: Strong immunoreactivity to D-β-aspartic acid-containing peptides was detected in the subepithelial amorphous materials of all surgical specimens with pingueculae. In contrast, no immunoreactivity to D-β-aspartic acid-containing peptides was detected in the specimens without pingueculae.Conclusions: Pingueculae are thought to be aggregates of proteins that contain D-β-aspartic acid residues. It is known that the conversion of L- to D-aspartyl residues is accelerated by ultraviolet irradiation. In addition, D-β-aspartic acid-containing proteins, in general, tend to aggregate with each other and accumulate in the tissues. These facts indicate that ultraviolet irradiation-induced racemisation of aspartic acid is closely related to the development of pingueculae

Glutaraldehyde is an effective cross-linker for production of antibodies against advanced glycation end-products

ArticleJOURNAL OF IMMUNOLOGICAL METHODS. 334(1-2): 82-90 (2008)journal articl

Advanced glycation end products (AGEs) promote melanogenesis through receptor for AGEs

Accumulation of advanced glycation end products (AGEs) is linked with development or aggravation of many degenerative processes or disorders, including aging and atherosclerosis. AGEs production in skin cells is known to promote stiffness and loss of elasticity through their buildup in connective tissue. However, the impact of AGEs has yet to be fully explored in melanocytes. In this study, we confirmed the existence of receptor for AGE (RAGE) in melanocytes in western blot and immunofluorescence along with increased melanin production in ex vivo skin organ culture and in vitro melanocyte culture following AGEs treatment. Cyclic AMP response element-binding protein (CREB) and extracellular signal-regulated kinases (ERK) 1/2 are considered as key regulatory proteins in AGEs-induced melanogenesis. In addition, blockage experiment using anti-RAGE blocking antibody has indicated that RAGE plays a pivotal role in AGE-mediated melanogenesis. Therefore, it is apparent that AGEs, known markers of aging, promote melanogenesis via RAGE. In addition, AGEs could be implicated in pigmentation associated with photoaging according to the results of increased secretion of AGEs from keratinocytes following UV irradiation. AGE-mediated melanogenesis may thus hold promise as a novel mean of altering skin pigmentationope

Tissue engineering strategies to bioengineer the ageing skin phenotype in vitro

Human skin ageing is a complex and heterogeneous process, which is influenced by genetically determined intrinsic factors and accelerated by cumulative exposure to extrinsic stressors. In the current world ageing demographic, there is a requirement for a bioengineered ageing skin model, to further the understanding of the intricate molecular mechanisms of skin ageing, and provide a distinct and biologically relevant platform for testing actives and formulations. There have been many recent advances in the development of skin models that recapitulate aspects of the ageing phenotype in vitro. This review encompasses the features of skin ageing, the molecular mechanisms that drive the ageing phenotype, and tissue engineering strategies that have been utilised to bioengineer ageing skin in vitro